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ABSTRACT
A method was developed for determining routinely the total pentosan content of wheat flour,
commercial B-starch and gluten. In this method, the polysaccharides in the sample were hydrolysed
by treatment with 2 HCl at 100°C. The resulting pentosan monosaccharides were characterised
by anion-exchange chromatography (HPAEC) combined with pulsed amperometric detection (PAD).
Using this method, water-extractable pentosans can be determined with only minor adjustments.
Interference by the relatively high concentration of starch-derived glucose in the sample, in the
HPAEC separation of the pentosan monosaccharides, was eliminated by enzymic conversion of
glucose into gluconic acid using glucose oxidase (GOD). Efficient removal of glucose (1 h incubation
time) required a relatively high amount of GOD and a continuous supply of oxygen into the solution.
The activity of GOD against galactose and xylose was subsequently blocked by lowering the pH of
the sample to about 1. The method described has good repeatability. The standard deviation in the
pentosan content (sum of the pentose sugars arabinose and xylose) was less than 0·1% for flour, B-
starch (pentosan content about 2%) and gluten (pentosan content of about 1%).
1997 Academic Press Limited
can also be determined by liquid chromatography in the tube was not removed. Following hydrolysis,
following hydrolysis12 or by gas chromatography, the sample was cooled and subsequently neu-
following hydrolysis and derivatisation to alditol tralised by addition of 4·0 NaOH (2·0 mL). Excess
acetates13 or to trimethylsilyl esters14. To prevent glucose was removed by addition of glucose ox-
interference of glucose, starch can be enzymically idase/catalase solution (2·0 mL) in 2·0 acetate
removed prior to hydrolysis12,13. buffer and 60 min incubation at room temperature.
In our study, a method was developed for de- During incubation the sample was mixed con-
termining routinely the pentosan content in wheat tinuously with the air present in the tube to en-
flour, commercial B-starch and gluten by anion- hance dissolution of oxygen. Finally the pH of the
exchange chromatography (HPAEC) with pulsed solution was lowered by addition of 4·0 HCl
amperometric detection (PAD). In this method, (1·0 mL) and an aliquot (0·5 mL) of internal stand-
the interference of glucose was eliminated by con- ard was added (1 mg/mL of 2-deoxy-D-galactose
version of glucose to gluconic acid with the enzyme in 0·04 HCl).
glucose oxidase (GOD). The method of analysis The amount of water-extractable pentosan can
is advantageous, because sample pretreatment is also be determined; this requires only minor ad-
simple and no derivatisation is required while the justments. The sample (ca. 0·4 g) was extracted
method gives both quantitative and qualitative with water (8·0 mL) for 2 h at 30°C2. After cen-
information about pentosan. trifugation, an aliquot (2·0 mL) of the supernatant
was removed, 4·0 HCl (2·0 mL) was added and
MATERIALS AND METHODS the sample was hydrolysed as described before.
Chemicals
Sample pretreatment was performed using hy- High performance anion-exchange
drochloric acid (Merck, Germany) and 50% (w/ chromatography
w) sodium hydroxide (Baker, Netherlands). For
the removal of glucose, the enzymes glucose ox- Samples were injected using a Marathon auto-
idase (EC 1.1.3.4, grade II, Boehringer, Germany) sampler (Spark, Holland) with a Tefzel rotor and
and catalase (EC 1.11.1.6, Sigma, U.S.A.) were a 20 lL loop. The pumps were obtained from
used, dissolved in buffer at 1000 U/mL and 6500 Gilson (France). The monosaccharides were sep-
U/mL, respectively. The buffer solutions were arated using a CarboPac PA1 precolumn and
prepared from sodium acetate (Merck) and acetic a CarboPac PA1 analytical column (Dionex,
acid (Merck) or tris(hydroxymethyl)-amino- U.S.A.). The monosaccharides were separated iso-
methane (Merck) and hydrochloric acid (Merck), cratically with 0·001 NaOH (45 min, flowrate
respectively. The following monosaccharides were 1 mL/min). Afterwards, strongly retained anions
used: galactose (Aldrich, Germany), glucose and were removed from the column by increasing the
1,5-gluconolactone (BDH, U.K.), xylose (Merck) NaOH concentration to 0·5 over a period of
and arabinose and 2-deoxy-D-galactose (Sigma). 6 min and continuing the elution with 0·5 NaOH
Wheat flour (Friso) was obtained from a local for a further 6 min. Finally, the NaOH con-
supermarket, while wheat B-starch (Puramyl VEM centration was reduced to 0·001 NaOH over a
II) was obtained from Latenstein (Netherlands). period of 1 min and the column was re-equilibrated
Wheat gluten was fractionated in our institute on with 0·001 NaOH for 7 min (flour and B-starch)
a laboratory scale. or for 4 min (gluten) before injecting another
sample. To maintain a high detector sensitivity,
0·25 NaOH was added to the effluent post-
Sample preparation prior to
column at a flow rate of 0·4 mL/min.
HPAEC/PAD analysis Monosaccharides were detected using a Dionex
For determination of the total pentosan content, PED I detector with a 3 mm gold working elec-
the sample (ca. 0·05 g) was dispersed in water trode. The potential of the electrode was pro-
(2·0 mL) and 4·0 HCl (2·0 mL) and then hy- grammed from +0·15 V (t = 0–0·72 s) to +0·7
drolysed for 90 min at 100°C in a 50 mL screw- V (t = 0·73–0·84 s) and finally −0·3 V (t =
cap tube. During hydrolysis, the samples were 0·85–1·2 s), while integrating the resulting signal
mixed frequently to aid dispersion. The air present from t = 0·52–0·72 s.
Determination of pentosan by HPAEC/PAD 39
2.50
2.00
Sugar content (% w/w)
1.50
1.00
0.50
Figure 1 Content (weight % of the sample as is) of arabinose (+), galactose (Α) and xylose (Β) and sum of the pentose
sugars arabinose and xylose (Φ) – as a function of the time of hydrolysis. The wheat flour sample was hydrolysed in 2 HCl,
treated with GOD and analysed by HPAEC/PAD.
100
80
60
Recovery (%)
40
20
Figure 2 Recovery (%) of arabinose (+), galactose (Α), xylose (Β) and 2-deoxy-D-galactose (Φ) after hydrolysis and analysis
by HPAEC/PAD. The recovery is defined as the concentration of the monosaccharides determined after acid treatment
divided by the concentration of the mixture without acid treatment.
52.84
800.00
600.00
Integrator signal (mV)
400.00
4
23.62
200.00
1
2
5
9.61
14.92
3
28.48
20.17
0.00
0 10 20 30 40 50 60
Time (min)
Figure 3 HPAEC/PAD chromatogram of a wheat flour sample after hydrolysis and GOD-treatment. The chromatographic
peaks can be assigned to (1) 2-deoxy-D-galactose (internal standard), (2) arabinose, (3) galactose, (4) glucose, (5) xylose and (6)
gluconic acid.
as those described in the literature by Englyst et consumed18. At a high glucose concentration, the
al.12,13, no time-consuming sample pre-treatment is level of available oxygen may be limiting. This
required consisting of enzymic starch degradation can, in part, be overcome by adding catalase which
followed by precipitation of pentosans with al- converts the hydrogen peroxide formed during the
cohol. GOD reaction into water and oxygen. Fur-
In the enzymic conversion of b-D-glucose, D- thermore, as a result of adding catalase, inhibition
gluconic acid is formed and molecular oxygen is of GOD by hydrogen peroxide is prevented. How-
42 R. Houben et al.
100
90
80
Degree of conversion (%)
70
60
50
40
15 30 45 60
Incubation time (min)
Figure 4 Degree of conversion of glucose (100 mg) when incubated with, respectively, 8000 U (+), 6000 U (Α), 4000 U
(Β) and 2000 U (Φ) of GOD in a large volume tube with a constant supply of air. When the sample was incubated in a
small volume tube (with 8000 U GOD), oxygen deficiency occurred (Η).
ever, only half the amount of oxygen consumed approximately 540 mg of glucose/min at pH 8·0
in the GOD conversion of glucose can be regained. (according to specifications).
We concluded that oxygen may become limiting
if incubation is carried out in closed tubes (Fig.
Quantification of the pentosan content
4). The oxygen consumed in the conversion of
glucose can be replenished readily, however, by A well-known problem in electrochemical de-
incubating in screw cap tubes with a relatively tection is the relative instability of the detector
large volume (4 mL of sample in a 50 mL tube), due to electrode fouling. This is partly overcome
continuously mixing the solution with air present in pulsed amperometric detection by programming
in the tube and frequently opening the tube to the potential of the electrode in a continuous way19.
refresh the air present. Nevertheless, detector sensitivity may change a
The rate of glucose conversion with GOD was little in the course of time, and this necessitates
much lower than expected. In a control ex- the use of an internal standard. In our study, we
periment, glucose (0·1 g) in Tris-HCl buffer, pH tested the sugars 2-deoxy-D-glucose, fucose and 2-
8·0 was treated with different amounts of GOD deoxy-D-galactose as internal standard. We found
and for different incubation times. As can be seen that 2-deoxy-D-glucose was not suitable because
in Figure 4, complete conversion of glucose within it was not well resolved from rhamnose, which
60 min required more than 4000 U of GOD. may be present in the sample in a low con-
This amount of enzyme, however, should convert centration. Fucose was not suitable because its
Determination of pentosan by HPAEC/PAD 43
Incubation deoxygal (%) ara (%) gal (%) glc (%) xyl (%)
time (h)
1 101 101 98 5 97
2 98 100 93 2 92
3 97 99 90 0 90
4 96 98 84 0 87
24 98 101 32 0 44
The concentration of the monosaccharides in the test mixture (about 50 mg/L) and the
amount of GOD added (2000 U) were comparable to the conditions described in the Materials
and Methods section.
chromatographic peak coincides partly with PAD- preserve the sample for a relatively long period of
sensitive compounds in the sample solution time. In this way, no (enzymic or acid) degradation
(e.g. Tris-buffer). These compounds temporarily of pentosan monosaccharides was observed over
lowered the detector sensitivity and thus interfered a time period of 7 days.
with the quantification of the internal standard.
2-Deoxy-D-galactose was finally chosen as the
internal standard because this sugar does not occur
naturally in wheat and it was well resolved from Determination of the pentosan content
pentosan monosaccharides (Fig. 3). It was added of wheat gluten
after hydrolysis because of the rapid degradation For the determination of the pentosan content of
of this sugar under acidic conditions (Fig. 2). wheat gluten, a small adjustment of the method
was necessary. As can be seen in Figure 5(a),
the detector signal was temporarily diminished
Substrate specificity of GOD
between retention times of about 6 and 9 min,
GOD has high specificity for b-D-glucose, but also probably due to a component resulting from pro-
catalyses the oxidation of galactose and xylose tein hydrolysis. This problem was negligible for
(relative rate about 100 times lower)18. The GOD wheat flour and wheat B-starch because of a much
incubation of the hydrolysed wheat sample should lower protein content. The disturbance in the
therefore be as short as possible, while nevertheless chromatographic signal partly coincided with the
ensuring almost complete conversion of glucose. chromatographic peak of 2-deoxy-D-galactose and
The effect of GOD on the pentosan sugars was thus hampered accurate quantification of the in-
determined by treating a mixture of mono- ternal standard. This interference led to a sig-
saccharides with GOD under the same conditions nificant over-estimation of the pentosan content.
as wheat samples (acetate buffer, pH=5) for time The problem was overcome by a small change
periods ranging from 1 to 24 h. The recoveries of in the chromatographic conditions, namely by
monosaccharides after GOD treatment are pre- lowering the interval between two injections from
sented in Table I. 7 to 4 min in combination with 1:1 dilution of the
From Table I it can be concluded that GOD sample with water prior to HPAEC/PAD analysis
catalyses the oxidation not only of glucose but also [Fig. 5(b)].
of galactose and xylose, whilst arabinose and 2- The recovery of arabinose and galactose, added
deoxy-D-galactose (internal standard) are hardly to the gluten matrix, and subsequently hydrolysed
affected. Treatment with GOD for 1 h resulted in with 2 HCl and analysed by HPAEC/PAD was
almost complete conversion of glucose to gluconic comparable to the recovery for a standard of
acid with negligible oxidation of pentosan mono- monosaccharides without sample. The recovery
saccharides. Subsequently, GOD activity needs to for xylose added to gluten, however, was about
be blocked by adding a small amount of HCl and 10% lower than when adding xylose either to
thus lowering the pH of the sample to about 1, to wheat flour or to wheat B-starch, implying that
44 R. Houben et al.
500.00
400.00
Integrator signal (mV)
3
300.00 1 2 18.97 4
9.08
14.10
22.17
5
26.44
200.00
100.00
(a)
0 5 10 15 20 25 30 35 40 45 50
Time (min)
500.00
400.00
Integrator signal (mV)
300.00 1
8.89
2 3
13.51
4
18.45
5
21.44
200.00
25.60
100.00
(b)
0 5 10 15 20 25 30 35 40 45 50
Time (min)
Determination of pentosan by HPAEC/PAD 45
Table II Standard deviation for the repeatability (sr) calculated from one-way analysis
of variance (ANOVA) from duplicate measurements on eight different days
The average contents of arabinose (ara), galactose (gal), xylose (xyl) and pentosan
(sum of pentose sugars arabinose and xylose multiplied by 0·88 to correct for anhydro
monosaccharides) are presented as weight % of the sample (as is).
xylose may be partly protected by the flour or B- resulting pentosan monosaccharides by anion-ex-
starch matrix. change chromatography (HPAEC) combined with
pulsed amperometric detection (PAD). The in-
terference of starch-derived glucose in the
Repeatability of the presented method HPAEC/PAD analysis can be eliminated simply
of analysis by enzymic conversion of glucose to gluconic acid
The standard deviation for the repeatability was with glucose oxidase. Following conversion of gluc-
determined by analysing the three sample matrices ose, the enzyme activity is blocked by lowering
in duplicate on eight different days. Table II the pH of the sample to about 1. The proposed
presents the standard deviation for the re- method for routine determination of the pentosan
peatability, as calculated by one-way analysis of content has good repeatability.
variance (ANOVA), and the calculated average
content. The standard deviation for the individual
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